Gli autori non hanno dichiarato alcun conflitto di interessi.

Il protocollo generale richiede 2 giorni, inclusi il primo giorno di induzione di sepsi e l'isolamento del liquido di BAL dall'animale e il secondo giorno per l'isolamento di esosomi dal mouse BALF. Tutte le procedure sono state esaminate e approvate dal comitato di uso Atlanta VA medical center e istituzionali Animal Care.
1. Mouse polmone settica acuta lesioni modello
<ol><li>Utilizzare 6-8 settimane - topi di C57BL/6J wild-type maschi vecchi (peso g 20-22) per l'animale modello ed eseguire tutte le procedure usando tecniche asettiche e strumenti. Autoclave tutti gli strumenti chirurgici, tra cui pinze e le forbici per 20 minuti a 121 ° C.</li>
	<li>Distribuire in modo casuale topi in sperimentale e gruppo di controllo. Trattenga i topi su una tavola chirurgica. Trattamento di topi con il lipopolysaccharide di Escherichia coli (LPS, 15 mg/kg) per via intraperitoneale. Secondo il peso corporeo individuale, diluire appropriati dosaggi di LPS a 0,1 mL di PBS per topo, quindi iniettare la soluzione di LPS usando una siringa da 1 mL con ago 27G e iniettare un volume equivalente di PBS agli animali di controllo.</li>
	<li>Tenere topi nella struttura BSL2 animale, che consiste di gabbie pulite a temperatura appropriata.</li>
	<li>24 ore più tardi, eutanasia topi con inalazione di CO2 .</li>
	<li>Selezionare l'area chirurgica nel centro del collo del mouse. Praticare una piccola incisione (circa 5 mm) e separare delicatamente i muscoli per l'accesso alla trachea con forcipe smussato fino a quando la trachea è esposto.</li>
	<li>Ritirare il liquido di BAL del mouse con siringa sterile da 10 mL con un ago da 27 G. In primo luogo, delicatamente, inserire l'ago nella trachea e iniettare 1 mL di PBS sterile lentamente nella trachea, quindi disegnare BALF in una siringa monouso da 3 mL con ago 27G. Al termine della procedura, smaltire siringhe e aghi in contenitore di sharps di biohazard.</li>
	<li>Trattare gli strumenti chirurgici con biossido di cloro, come descritto in precedenza da Chauret et al. 27e quindi sterilizzare tutti gli strumenti chirurgici in autoclave per 20 minuti a 121 ° C.</li>
</ol>2. esosomi isolamento-serie Ultra-centrifugazione e caratterizzazione
<ol><li>Raccogliere il liquido di BAL del mouse in provette coniche da 15 mL. Per raccogliere le cellule alveolari, centrifugare i campioni per 10 min a 1.000 g, a 4 ° C.</li>
	<li>Trasferire il surnatante nuove provette coniche da 15 mL, quindi centrifugare i campioni per 30 min a 4.000 x g a 4 ° C per rimuovere i detriti cellulari.</li>
	<li>Raccogliere il surnatante in ultracentrifuga tubi e centrifugare i campioni per 60 min a 10.000 x g a 4 ° C per rimuovere le particelle più grandi di cellulare.</li>
	<li>Trasferire il surnatante in nuove provette coniche, quindi filtrare i campioni attraverso 0,2 µm siringa-filtro per rimuovere i restanti particelle più grandi.</li>
	<li>Raccogliere campioni in tubi ultracentrifuga e l'equilibrio di tubi. Centrifugare i campioni per 2 h a 140.000 x g a 4° C a pellet gli esosomi. Nota: La dimensione di esosomi varia 20-200 nanometro. 0,2 µm filtro viene utilizzato per la rimozione di particelle più grandi che sono più di 200 nm in dimensione.</li>
	<li>Scartare i surnatanti delicatamente. Dissolva la pallina con 100 µ l PBS o soluzione di lisi e trasferimento esosomi campioni per provette da 1,5 mL e conservare a-80 ° C fino all'utilizzo. Nota: Microscopio elettronico a trasmissione è consigliabile verificare gli esosomi isolati.</li>
	<li>Seguire il campione TEM standard elaborazione protocollo28. Analisi di campioni di esosomi usando microscopia elettronica di trasmissione e prendere immagini, scala bar 200 nm.</li>
</ol>3. preparazione del miRNA e Performing miRNA-q-RT-PCR
Nota: miRNA-q-RT-PCR si raccomanda di analizzare il livello di espressione dei miRNA.
<ol><li>Seguire il protocollo di isolamento standard Mirna e assicurarsi di utilizzare una quantità uguale di RNA purificato nella trascrizione inversa single-strand24.</li>
	<li>Seguire il protocollo standard di PCR in tempo reale per rilevare il livello di microRNA exosomal in liquido di BAL. Utilizzare specifici iniettori di PCR in tempo reale contro miR-155, miR-146a e SnRNA U6. Eseguire la reazione di PCR con apparato PCR quantitativa e normalizzare i valori di espressione per controllo interno U6 snRNA. Nota: Ogni gruppo è costituito da campioni triplici24.</li>
</ol>4. esosomi etichettatura e purificazione
<ol><li>Sospendere gli esosomi pellet con 100 µ l di soluzione di sospensione in una provetta di polipropilene da 1,5 mL, quindi aggiungere 0,4 µ l della tintura esosomi nell'altro tubo con 100 µ l della soluzione di sospensione.</li>
	<li>Aggiungere esosomi nella soluzione per la soluzione colorante e mescolare accuratamente pipettando rapidamente. Conservare i campioni per 5 min a temperatura ambiente, proteggere dalla luce.</li>
	<li>Mettere una colonna di esosomi spin in una provetta di eluizione da 1,5 mL. Trasferire la miscela di soluzione al centro della colonna di spin di esosomi.</li>
	<li>Girare la colonna per 2 min a 800 x g a temperatura ambiente.</li>
	<li>Scartare la colonna e conservare il campione eluito a-20 ° C fino all'utilizzo.</li>
</ol>5. convalida di esosomi Up preso da cellule recettive
<ol><li>Semi di 1 x 104 MLE 12 prodotto in una diapositiva di 8 pozzetti camera e cellule di coltura a 37 ° C, 5% CO2.</li>
	<li>Aggiungere 10 µ l con etichetta fluorescente esosomi alle celle. Incubare per 1 h a 37 ° C.</li>
	<li>Rimuovere i mezzi di coltura e lavare le cellule con PBS, quindi fissare le cellule con paraformaldeide 1% per 10 minuti a temperatura ambiente.</li>
	<li>Montare il vetrino in un mezzo di montaggio con DAPI.</li>
	<li>Analizzare i campioni attraverso un microscopio a fluorescenza e prendere le immagini, ingrandimento 40x.</li>
</ol>6. Verifica dell'interazione tra esosomi e cellule recettive
<ol><li>Seme 2 x 105 MLE12/pozzetto di una piastra di coltura 12-pozzetti contenenti terreno di coltura appropriato e cellule di coltura a 37 ° C, 5% CO2.</li>
	<li>Aggiungere 20 µ g esosomi dal fluido del mouse BAL MLE12 celle e impostare ripetizioni per gruppo sperimentale.</li>
	<li>24 ore dopo, lavare le cellule con PBS e aggiungere 600 µ l di tampone di lisi in pozzetto. Agitare la piastra delicatamente e raccogliere campioni di lisi in provetta da 1,5 mL con la pipetta.</li>
	<li>Seguire il protocollo di isolamento di RNA totale standard e protocollo di trascrizione d'inversione del cDNA, completare la trascrizione inversa del cDNA del singolo-filo29.</li>
	<li>Seguire il protocollo standard di PCR in tempo reale per rilevare il livello di espressione di mRNA in cellule recettive29. Utilizzare specifici iniettori di PCR in tempo reale contro TNF-α, IL-6 e ZO-1. Eseguire reazioni di PCR con apparato PCR quantitativa e normalizzare i valori di espressione per il controllo interno GAPDH. Nota: Ogni gruppo è costituito da campioni triplici.</li>
</ol>

<ol>
	<li>Rubenfeld, G. D., Herridge, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+outcomes+of+acute+lung+injury.">Epidemiology and outcomes of acute lung injury.</a> Chest. 131, 554-562 (2007).</li><li>Baron, R. M., Choi, A. J., Owen, C. A., Choi, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+manipulated+mouse+models+of+lung+disease:+potential+and+pitfalls.">Genetically manipulated mouse models of lung disease: potential and pitfalls.</a> American journal of physiology. Lung cellular and molecular physiology. , L485-L497 (2012).</li><li>Han, S., Mallampalli, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome:+from+mechanism+to+translation.">The acute respiratory distress syndrome: from mechanism to translation.</a> Journal of immunology. , 855-860 (1950).</li><li>Aeffner, F., Bolon, B., Davis, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+Acute+Respiratory+Distress+Syndrome:+A+Review+of+Analytical+Approaches,+Pathologic+Features,+and+Common+Measurements.">Mouse Models of Acute Respiratory Distress Syndrome: A Review of Analytical Approaches, Pathologic Features, and Common Measurements.</a> Toxicologic pathology. 43, 1074-1092 (2015).</li><li>Matute-Bello, G., Frevert, C. W., Martin, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+acute+lung+injury.">Animal models of acute lung injury.</a> American journal of physiology. Lung cellular and molecular physiology. , L379-L399 (2008).</li><li>Terrasini, N., Lionetti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+in+Critical+Illness.">Exosomes in Critical Illness.</a> Critical care medicine. , (2017).</li><li>Fujita, Y., Kadota, T., Araya, J., Ochiya, T., Kuwano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles:+New+Players+in+Lung+Immunity.">Extracellular Vesicles: New Players in Lung Immunity.</a> American journal of respiratory cell and molecular biology. , (2017).</li><li>Kubo, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles+in+Lung+Disease.">Extracellular Vesicles in Lung Disease.</a> Chest. 153, 210-216 (2018).</li><li>Krol, J., Loedige, I., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+widespread+regulation+of+microRNA+biogenesis,+function+and+decay.">The widespread regulation of microRNA biogenesis, function and decay.</a> Nature reviews. Genetics. 11, 597-610 (2010).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+target+recognition+and+regulatory+functions.">MicroRNAs: target recognition and regulatory functions.</a> Cell. 136, 215-233 (2009).</li><li>Fanini, F., Fabbri, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CANCER-DERIVED+EXOSOMIC+microRNAs+SHAPE+THE+IMMUNE+SYSTEM+WITHIN+THE+TUMOR+MICROENVIRONMENT:+STATE+OF+THE+ART.">CANCER-DERIVED EXOSOMIC microRNAs SHAPE THE IMMUNE SYSTEM WITHIN THE TUMOR MICROENVIRONMENT: STATE OF THE ART.</a> Seminars in cell &amp; developmental biology. , (2016).</li><li>Agarwal, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental,+Systems,+and+Computational+Approaches+to+Understanding+the+MicroRNA-Mediated+Reparative+Potential+of+Cardiac+Progenitor+Cell-Derived+Exosomes+From+Pediatric+Patients.">Experimental, Systems, and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients.</a> Circulation research. 120, 701-712 (2017).</li><li>Prattichizzo, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+microRNAs+and+endothelial+hyperglycaemic+memory:+a+therapeutic+opportunity?.">Extracellular microRNAs and endothelial hyperglycaemic memory: a therapeutic opportunity?.</a> Diabetes, obesity &amp; metabolism. 18, 855-867 (2016).</li><li>Huang-Doran, I., Zhang, C. Y., Vidal-Puig, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles:+Novel+Mediators+of+Cell+Communication+In+Metabolic+Disease.">Extracellular Vesicles: Novel Mediators of Cell Communication In Metabolic Disease.</a> Trends in endocrinology and metabolism: TEM. 28, 3-18 (2017).</li><li>Khalyfa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Plasma+Extracellular+Microvesicle+MicroRNA+Cargo+and+Endothelial+Dysfunction+in+Children+with+Obstructive+Sleep+Apnea.">Circulating Plasma Extracellular Microvesicle MicroRNA Cargo and Endothelial Dysfunction in Children with Obstructive Sleep Apnea.</a> American journal of respiratory and critical care medicine. 194, 1116-1126 (2016).</li><li>Thomou, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+circulating+miRNAs+regulate+gene+expression+in+other+tissues.">Adipose-derived circulating miRNAs regulate gene expression in other tissues.</a> Nature. 542, 450-455 (2017).</li><li>Gregson, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+Exosomal+RNA+Profiles+in+Bronchoalveolar+Lavage+from+Lung+Transplants+with+Acute+Rejection.">Altered Exosomal RNA Profiles in Bronchoalveolar Lavage from Lung Transplants with Acute Rejection.</a> American journal of respiratory and critical care medicine. 192, 1490-1503 (2015).</li><li>Valadi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-mediated+transfer+of+mRNAs+and+microRNAs+is+a+novel+mechanism+of+genetic+exchange+between+cells.">Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells.</a> Nature cell biology. 9, 654-659 (2007).</li><li>Doxaki, C., Kampranis, S. C., Eliopoulos, A. G., Spilianakis, C., Tsatsanis, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinated+Regulation+of+miR-155+and+miR-146a+Genes+during+Induction+of+Endotoxin+Tolerance+in+Macrophages.">Coordinated Regulation of miR-155 and miR-146a Genes during Induction of Endotoxin Tolerance in Macrophages.</a> Journal of immunology. , 5750-5761 (2015).</li><li>Schulte, L. N., Westermann, A. J., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+activation+and+functional+specialization+of+miR-146+and+miR-155+in+innate+immune+sensing.">Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing.</a> Nucleic acids research. 41, 542-553 (2013).</li><li>Alexander, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome-delivered+microRNAs+modulate+the+inflammatory+response+to+endotoxin.">Exosome-delivered microRNAs modulate the inflammatory response to endotoxin.</a> Nature communications. 6, 7321 (2015).</li><li>Yuan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1+is+induced+in+tumor+associated+macrophages+by+cyclo-oxygenase+pathway+in+human+non-small+cell+lung+cancer.">TREM-1 is induced in tumor associated macrophages by cyclo-oxygenase pathway in human non-small cell lung cancer.</a> PloS one. 9, e94241 (2014).</li><li>Xu, R., Richards, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+In+Vitro+Co-Culture+Model+in+Anti-Cancer+Drug+Development+Cascade.">Development of In Vitro Co-Culture Model in Anti-Cancer Drug Development Cascade.</a> Combinatorial chemistry &amp; high throughput screening. 20, 451-457 (2017).</li><li>Yuan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TREM-1-accentuated+lung+injury+via+miR-155+is+inhibited+by+LP17+nanomedicine.">TREM-1-accentuated lung injury via miR-155 is inhibited by LP17 nanomedicine.</a> American journal of physiology. Lung cellular and molecular physiology. , L426-L438 (2016).</li><li>Sadikot, R. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-dose+dexamethasone+accentuates+nuclear+factor-kappa+b+activation+in+endotoxin-treated+mice.">High-dose dexamethasone accentuates nuclear factor-kappa b activation in endotoxin-treated mice.</a> American journal of respiratory and critical care medicine. 164, 873-878 (2001).</li><li>Yuan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin+mediated+epigenetic+modulation+inhibits+TREM-1+expression+in+response+to+lipopolysaccharide.">Curcumin mediated epigenetic modulation inhibits TREM-1 expression in response to lipopolysaccharide.</a> The international journal of biochemistry &amp; cell biology. 44, 2032-2043 (2012).</li><li>Chauret, C. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chlorine+dioxide+inactivation+of+Cryptosporidium+parvum+oocysts+and+bacterial+spore+indicators.">Chlorine dioxide inactivation of Cryptosporidium parvum oocysts and bacterial spore indicators.</a> Applied and environmental microbiology. 67, 2993-3001 (2001).</li><li>Cizmar, P., Yuana, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+Characterization+of+Extracellular+Vesicles+by+Transmission+and+Cryo-Transmission+Electron+Microscopy.">Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy.</a> Methods in molecular biology. , 221-232 (2017).</li><li>Yuan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-related+proteins+prolong+macrophage+survival+through+induction+of+Triggering+receptor+expressed+on+myeloid+cells-1.">HIV-related proteins prolong macrophage survival through induction of Triggering receptor expressed on myeloid cells-1.</a> Scientific reports. 7, 42028 (2017).</li><li>Jackson, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Mitochondrial+Transfer+in+Direct+Co-cultures+of+Human+Monocyte-derived+Macrophages+(MDM)+and+Mesenchymal+Stem+Cells+(MSC).">Analysis of Mitochondrial Transfer in Direct Co-cultures of Human Monocyte-derived Macrophages (MDM) and Mesenchymal Stem Cells (MSC).</a> Bio-protocol. 7, (2017).</li><li>Fujita, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Vesicles:+New+Players+in+Lung+Immunity.">Extracellular Vesicles: New Players in Lung Immunity.</a> American journal of respiratory cell and molecular biology. 17, 474-484 (2017).</li><li>Gunasekaran, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-Derived+Exosomes+With+Lung+Self-Antigens+in+Human+Lung+Allograft+Rejection.">Donor-Derived Exosomes With Lung Self-Antigens in Human Lung Allograft Rejection.</a> American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons. 17, 474-484 (2017).</li><li>Kalra, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+evaluation+of+plasma+exosome+isolation+techniques+and+assessment+of+the+stability+of+exosomes+in+normal+human+blood+plasma.">Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.</a> Proteomics. 13, 3354-3364 (2013).</li><li>Baranyai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+Exosomes+from+Blood+Plasma:+Qualitative+and+Quantitative+Comparison+of+Ultracentrifugation+and+Size+Exclusion+Chromatography+Methods.">Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.</a> PloS one. 10, e0145686 (2015).</li><li>Nordin, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafiltration+with+size-exclusion+liquid+chromatography+for+high+yield+isolation+of+extracellular+vesicles+preserving+intact+biophysical+and+functional+properties.">Ultrafiltration with size-exclusion liquid chromatography for high yield isolation of extracellular vesicles preserving intact biophysical and functional properties.</a> Nanomedicine : nanotechnology, biology, and medicine. 11, 879-883 (2015).</li></ol>

Questo lavoro è stato supportato da recensione di merito VA 2I01BX001786-06A1 finanziamenti a RS.

Modelli murini di malattie sono comunemente utilizzati per valutare la funzione fisiologica dei geni specifici e per ridurre il costo della sperimentazione2. La ferita di polmone acuta settica descritta qui imita risposta infiammatoria visto in esseri umani con ARDS. Questo modello è rilevante per studiare la patogenesi molecolare, sviluppo di biomarcatori e per testare potenziali nuove terapie5.
Sistemi di co-coltura sono rilevanti per gli stud...

ALI e ARDS sono le forme pericolose di insufficienza respiratoria con ipossiemia severa causata da edema polmonare non cardiogeno che colpisce circa 1 milione di persone nel mondo ogni anno1. L'eziologia di ARDS include ferita diretta ai polmoni da infezioni o aspirazione e una varietà di insulti indiretti. Nell'ultimo decennio c'è stata una maggiore comprensione della patogenesi molecolare di ARDS, tuttavia, trattamenti specifici mirati per ARDS sono ancora essere sviluppato2,3.
Sono stati sviluppati diversi modelli animali di danno polmonare acuto che forniscono un ponte per la traduzione di terapie sperimentali per studi umani4,5. I modelli comunemente usati includono installazione locale di acido oleico, batteri, LPS e bleomicina. Altri approcci includono ischemia-riperfusione, puntura cecal legatura, lesione elasticizzato indotta da ventilazione meccanica, l'iperossia o somministrazione sistemica di batteri e LPS5. Questi modelli forniscono un sistema biologico utile per testare le ipotesi cliniche e per lo sviluppo di potenziali terapie. Per simulare umano ARDS, modelli animali dovrebbero riprodurre l'infiammazione e lesione acuta alle cellule epiteliali ed endoteliali con difetti nella funzione di barriera nei polmoni.
Gli esosomi sono vescicole di membrana con 20-200 nanometro di diametro, il cui contenuto molecolare contiene proteine, DNA, RNA e lipidi, e facilitano la comunicazione intercellulare nel microambiente del tessuto tramite trasferimento composizione molecolare. Gli esosomi sono secreti da più tipi di cellule, quali cellule endoteliali, cellule epiteliali, cellule muscolari lisce e cellule del tumore e presenti nel fluido del corpo umano. Studi indicano che gli esosomi regolano cross-talk tra le cellule immunitarie e le cellule stromali durante malattie infiammatorie, infettive e sterile, e loro rilascio anormale sembra essere regolata da vari stimoli naturali e sperimentali durante fisiologico e processi patologici6. Tale rete di comunicazione può svolgere un ruolo importante nella patogenesi di malattie polmonari e può influenzare la progressione fisiopatologico7,8. Come 18-22 nucleotidi RNA non codificanti, Mirna esiste nel siero sia dei tessuti e fluidi corporei, al plasma e modula l'espressione di mRNA a livello post-traduzionale9,10.
Mirna confezionati in esosomi influenzano la differenziazione e la funzione di più tipi di cellule, e livelli eccessivi sono associati con una varietà di malattie, compreso il cancro, malattie polmonari, obesità, diabete e malattia cardiovascolare11, 12,13,14,15,16. Entrata in cellule del destinatario e la spola di Mirna exosomal facilitare comunicazioni intercellulari modificando l'emostasi del microambiente17,18. Ferita di polmone acuta è un processo complesso, che prevedono più tipi di cellule con vasta comunicazione intercellulare attraverso esosomi8. miR-155 e miR-146a condividere il comune meccanismo di regolazione trascrizionale e contribuire alla risposta infiammatoria e la tolleranza immunitaria19,20. Gli studi recenti indicano che entrambi modulano la risposta infiammatoria via exosomal Mirna spola tra cellule immunitarie21. Tuttavia, i meccanismi molecolari sottostanti effetti modulatory di Mirna exosomal alveolare risposta all'endotossina rimangono poco chiari, senza dubbio la potenziale rilevanza clinica e traslazionale implicazione merito ulteriore indagine.
Modelli di co-coltura sono impiegati per definire l'interazione dei tipi cellulari specifici nell'ambiente complesso, quali l'infiammazione e cancro22,23. Queste piattaforme forniscono una strategia alternativa per interrogare i cross-talk tra tipi cellulari in particolare per le cellule immuni e strutturali.
Endotracheale, amministrazione aerosolizzato, intraperitoneale o sistemica di LPS sono ampiamente usati per indurre sperimentale del polmone lesioni24,25,26e hanno dimostrato di indurre permeabilità epiteliale ed endoteliale difetti. Qui usiamo LPS intraperitoneale per indurre un modello settico della ferita acuta del polmone in topi. Entro 24 h dalla somministrazione intraperitoneale di LPS, difetti di permeabilità sono indotte nei polmoni con il reclutamento di cellule infiammatorie. Inoltre, mostriamo che esosomi da BAL contengono miRNA-155 e miR-146a, ed esosomi da liquido di BAL inducono l'espressione di citochine pro-infiammatorie in cellule epiteliali destinatario, compreso IL-6 e TNF-α. Questi dati sono il primo a mostrare che i miRNA exosomal vengono secreti nel BAL in questo modello di danno polmonare acuto settico.

<table border="0" cellpadding="0" cellspacing="0" style="width:1276px;" width="1276">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:691px;">lipopolysaccharide</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:224px;">Escherichia coli 055:B5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS pH7.2(1X)</td>
			<td>Life technologies</td>
			<td>20012-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mirVana miRNA isolation kit</td>
			<td>Thermo Fisher</td>
			<td align="right">449774</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>Thermo Fisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mmu-miR-155 (002571) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">has-miR-146a (000468) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">U6snRNA primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4427975</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TNF-&#945;(Mm00443258_m1 ) primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IL-6 (Mm00446190_m1&#160;&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZO-1 (Mm00493699_m1&#160; )primer</td>
			<td>Thermo Fisher</td>
			<td align="right">4331182</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">minimal essential medium (MEM)</td>
			<td>Thermo Fisher</td>
			<td>11095-072</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trysin-EDTA solution(0.05%)</td>
			<td>Thermo Fisher</td>
			<td align="right">25300054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosomes were labeled with PKH67 through PKH67 Green Fluorescent Cell linker Mini Kit&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>MINI67-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Exosome Spin Columns (MW3000)</td>
			<td>Thermo Fisher</td>
			<td align="right">4484449</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Beckman Coulter Optima L-100XP ultracentrifuge&#160;</td>
			<td>Beckman Coulter</td>
			<td>L-100XP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultra-clear centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td align="right">344058</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Applied Biosystems 7500 Fast Real-Time PCR System</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transmission electron microscopes (TEM)</td>
			<td>JEOL Ltd</td>
			<td>120 kV TEM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus BX41 microscope</td>
			<td>Olympus</td>
			<td>BX41</td>
			<td></td>
		</tr>
	</tbody>
</table>

bronchoalveolar lavage exosomes in lipopolysaccharide-induced septic lung injury

Lesioni polmonari acute (ALI) e sindrome da distress respiratorio (ARDS) rappresentano un gruppo eterogeneo di malattie polmonari che continua ad avere un'alta morbosità e mortalità. Patogenesi molecolare delle ALI è essere meglio definiti; Tuttavia, a causa della natura complessa della malattia terapie molecolari devono ancora essere sviluppato. Qui usiamo un modello di topo indotta lipopolysaccharide (LPS) di danno polmonare acuto settica di delineare il ruolo degli esosomi nella risposta infiammatoria. Utilizzando questo modello, siamo stati in grado di dimostrare che i topi che sono esposti a LPS intraperitoneali secernono esosomi nel fluido di lavaggio (BAL) di bronco-alveolare dai polmoni che sono confezionati con miRNA e citochine che regolano la risposta infiammatoria. Ulteriormente utilizzando un sistema di modello di co-coltura, mostriamo che gli esosomi rilasciati dai macrofagi interrompono espressione delle proteine di giunzione stretta in cellule epiteliali bronchiali. Questi risultati indicano che 1) cross-talk tra cellule innate immune e strutturali attraverso la marcia exosomal contribuiscono alla risposta infiammatoria e rottura della barriera strutturale e 2) targeting questi miRNA può fornire una nuova piattaforma per trattare ALI e ARDS.

Per indurre il danno polmonare settico, i topi sono stati trattati con LPS intraperitoneali (15 mg/kg). Entro 24 h dalla amministrazione dei LPS, neutrophilic afflusso è stato visto nei polmoni, come mostrato in Figura 1A. Liquido di BAL del mouse è stato disegnato, seguita da isolamento e purificazione di esosomi. Morfologia di BALF esosomi è stata confermata da microscopia elettronica di trasmissione (Figura 1B).
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Watch this Scientific Journal Video about Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury at JoVE.com

Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

Il lavaggio broncoalveolare esosomi in lesioni polmonari settici Lipopolysaccharide indotta

Topi esposti a LPS intraperitoneali secernono esosomi nel fluido di lavaggio (BAL) bronco-alveolare che sono confezionati con Mirna. Utilizzando un sistema di co-coltura, indichiamo che esosomi rilasciati nel liquido di BAL perturbare l'espressione delle proteine di giunzione stretta in cellule epiteliali bronchiali ed aumentano l'espressione delle citochine pro-infiammatorie che accentuano la ferita di polmone.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) represent a heterogeneous group of lung diseases which continues to have a high ...

bronchoalveolar-lavage-exosomes-lipopolysaccharide-induced-septic

Research

JoVE Journal

Genetics

10.8K Views.  Atlanta VAMC. Topi esposti a LPS intraperitoneali secernono esosomi nel fluido di lavaggio (BAL) bronco-alveolare che sono confezionati con Mirna. Utilizzando un sistema di co-coltura, indichiamo che esosomi rilasciati nel liquido di BAL perturbare l'espressione delle proteine di giunzione stretta in cellule epiteliali bronchiali ed aumentano l'espressione delle citochine pro-infiammatorie che accentuano la ferita di polmone.

Video: Bronchoalveolar Lavage Exosomes in Lipopolysaccharide-induced Septic Lung Injury

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

siRNA Transfection e l&#39;EMSA Analisi su appena isolate umani dei villi citotrofoblasti

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Ricerca

Genetica

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

In Situ etichettatura di DNA mitocondriale di replica in Drosophila adulti ovaie di Edu colorazione

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Optogenetic caso Mutagenesi Utilizzando istone-miniSOG in<em&gt; C. elegans</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Allevamento e RNA a doppio filamento mediata silenziamento genico nel Hide Beetle,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

In pool shRNA schermo per riattivazione di MeCP2 sul cromosoma di X inattivo

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly defined states of chromatin organization. The dynamic architecture of the nucleus is controlled by multiple mechanisms and shapes the transcriptional output programs. It is, therefore, important to determine locus-specific chromatin accessibility in a reliable fashion that is preferably independent from antibodies, which can be a potentially confounding source of experimental variability. Chromatin accessibility can be measured by various methods, including the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) assay, that allow the determination of general chromatin accessibility in a relatively low number of cells. Here we describe a FAIRE protocol that allows simple, reliable, and fast identification of genomic regions with a low protein occupancy. In this method, the DNA is covalently bound to the chromatin proteins using formaldehyde as a crosslinking agent and sheared to small pieces. The free DNA is afterwards enriched using phenol:chloroform extraction. The ratio of free DNA is determined by quantitative polymerase chain reaction (qPCR) or DNA sequencing (DNA-seq) compared to a control sample representing total DNA. The regions with a looser chromatin structure are enriched in the free DNA sample, thus allowing the identification of genomic regions with lower chromatin compaction.

The plasticity of nuclear architecture is controlled by dynamic epigenetic mechanisms including non-coding RNAs, DNA methylation, nucleosome repositioning as well as histone composition and modification. Here we describe a Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) protocol which allows the determination of chromatin accessibility in a reproducible, inexpensive, and straightforward manner.

Formaldeide-assistita di isolamento di elementi regolatori per l'accessibilità della cromatina di misura in cellule di mammifero

Appropriate gene expression in response to extracellular cues, that is, tissue- and lineage-specific gene transcription, critically depends on highly ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

CRISPR-mediata di riorganizzazione della struttura della cromatina Loop

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Identificazione di eventi di ricombinazione omologa in cellule staminali embrionali del Mouse mediante Southern Blotting e reazione a catena della polimerasi

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Alterazioni di modificazione post-traduzionale istone caratterizzante nel lievito Neurodegenerative Proteinopathy modelli

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

Determinare il tasso di fertilizzazione dell'uovo di Bemisia tabaci utilizzando una tecnica citogenetica

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...